The present invention is to provide a nucleic acid molecule encoding a cytochrome P450 hydroxylase that catalyzes the brassinosteroid biosynthesis in plants, the methods and processes for generating and analyzing biologically active polypeptides encoded by the nucleic acid molecule, and the identification and characterization of other signaling proteins that regulate the brassinosteroid biosynthesis.
Light regulates virtually all aspects of plant growth and developmental processes, among which seedling development is the most sensitive to light condition (Arnim and Deng, 1996; Chory, 2000). Plants therefore possess sophisticated systems for light signal perception and transmission. Light signals are perceived by various photoreceptors, including the red and far-red light absorbing phytochromes (Quail, 1997), the blue/UV-A light absorbing cryptochromes/phototropin (Briggs and Huala, 1999), and the UV-B light absorbing receptor (Senger and Schmidt, 1994). The light signals are subsequently transmitted through various signal transducers and finally regulate genes involved in plant photomorphogenesis. Light does not function independently but is integrated with endogenous growth regulators, such as growth hormones, for temporal and spatial regulation of growth and development (Szekeres et al., 1996; Schumacher and Chory, 2000).
Recent studies on photomorphogenic mutants suggest that brassinosteroids (BR), auxin, and gibberellins (GA) are involved in the photomorphogenic processes, particularly stem morphogenesis and leaf development (Li et al., 1996; Kim et al., 1998; Friedborg et al., 1999; Kamiya et al., 1999). Among them, the most extensively studied is the interaction between light and BR (Szekeres et al., 1996; Clouse and Sasse, 1998; Schumacher and Chory, 2000). BR-deficient mutants exhibit photomorphogenic development in the dark, such as chlorophyll synthesis, apical hook and cotyledon opening, and thick dwarfish hypocotyls (Fujioka et al., 1997). In the light they show dwarfish stems and petioles, dark-green leaves, male sterility, and delayed senescence, primarily due to retarded cell elongation in stems and pollen tubes (Li et al., 1996; Szekeres et al., 1996). These observations indicate that BR hormones possess an essential role in plant growth and developmental processes, including cell elongation and division, etiolation, reproductive development, and vascular differentiation (Clouse and Sasse, 1998).
BR hormones are synthesized through a multi-step biosynthetic pathway by a series of enzymes in plants. The biosynthetic steps have been elucidated using cultured cells and seedlings of Catharanthus roseus and by feeding experiments of BR-deficient mutants (Clouse and Sasse, 1998; Fujioka et al., 2000). The enzymes characterized so far include sterol desaturases (DWF7/STE1) (Choe et al., 1999b), oxidases (DWF1/DIM1/LKB) (Choe et al., 1999a; Nomura et al., 1999), reductases (DET2/LK) (Li et al., 1996), and cytochrome P450 hydroxylases (DWF4, CPD/DWF3, D) (Choe et al., 1998; Bishop et al., 1999). An Arabidopsis mutant bril and a pea mutant lka are insensitive to BR and have mutations in BR perception (Li and Chory, 1997; Nomura et al., 1999). The BRII gene encodes a leucine-rich repeat (LRR) receptor with the cytoplasmic serine/threonine kinase domain and the external putative BR binding LRR domain (Li and Chory, 1997). The external LRR domain has been recently confirmed to respond to BR (He et al., 2000). Several proteins, such as expansins (Cosgrove, 1997), endo-1,4-xcex2-D-glucanases (Nicol et al., 1998), and xyloglucan endotransglycosylase (Xu et al., 1995), respond to BR signals. Interestingly, the BR-responsive proteins have been implicated to be primarily responsible for the cell wall modification in the cell elongation and related process, which are primary developmental processes regulated by light and BR (Salchert et al., 1998; Azpiroz et al., 1998).
Roles of a variety of signaling mediators have been confirmed or suggested in light signal transduction pathway in plants, including guanosine triphosphatases (GTPases), Ca2+/calmodulin, phospholipase C, and protein kinases/phophatases (Roux, 1994). Heterotrimeric GTPases modulate the light signal transduction in plants through interaction with cGMP and/or Ca2+ (Bowler et al., 1994; Hooley, 1998). Monomeric small GTPases, another group of GTPases that belong to the Ras superfamily, regulate numerous cellular processes in animals and plants, such as cell growth and differentiation, cell morphogenesis, and vesicle transport (Ma, 1994; Exon, 1998). Accumulating evidences support that they also fulfill a role in the light signal transduction in plants (Romero et al., 1991; Sommer and Song, 1994; Nagano et al., 1995). Of particular interest is the pea Pra2 small GTPase. The expression is dark-inducible and down-regulated by the light (Yoshida et al., 1993). It is thus notable that the 5xe2x80x2 nontranslating region of the pra2 gene contains a dark-inducible element, DE1, that confers light down-regulation of a reporter gene (Inaba et al., 2000). The Pra2 is expressed exclusively in the rapidly elongating upper region of the epicotyls in the dark (Nagano et al., 1995). It is interesting that this plant part is the site where total phytochrome content is the richest among different plant parts (Briggs and Siegelman, 1965) and most sensitive to BR treatment in BR-deficient dwarfish mutants (Azpiroz et al., 1998). These observations propose that the Pra2 plays a regulatory role in the integration of light signals with plant growth hormones, most probably BR hormones, for the regulation of etiolated seedling development (Arnim and Deng, 1996).
In this work, we show that the Pra2 specifically interacts with a noble cytochrome P450 enzyme involved in the BR biosynthesis. The P450 is dark-inducible and predominantly expressed in the rapidly elongating region of the epicotyls, like the Pra2. The Pra2 and cytochrome P450 proteins are colocalized to endoplasmic reticulum (ER). Transgenic plants with reduced Pra2 exhibits dwarfish hypocotyls in the dark, which is completely rescued by BR but not by other growth hormones. The cytochrome P450 mediates multiple C-2 hydroxylations in the BR biosynthesis. Surprisingly, transgenic plants overexpressing the cytochrome P450 show elongated stems even in the light, which phenocopies the hypocotyls of dark-grown seedlings. These results indicate that the Pra2 is a light-regulated molecular switch that regulates the hypocotyl elongation in etiolated seedlings through interaction with the cytochrome P450. The Pra2-P450 interaction could be a molecular mechanism underlying the dark developmental process (etiolation) in plants.
The present invention relates to nucleic acid molecules encoding a cytochrome P450 hydroxylase or biologically active fragments of such a protein that catalyze the conversion from typhasterol to castasterone via C-2 hydroxylations in the brassinosteroid biosynthesis in plants. Such nucleic acid molecules preferentially encode a protein with the amino acid sequence as given in SEQ ID NO: 2 or fragments thereof that possess the enzymatic activity of the above-described cytochrome P450-like hydroxylase.
The present invention also relates to nucleic acid molecules that hybridize under high stringent conditions to a nucleic acid molecule as given in SEQ ID NO: 1. The term xe2x80x9chybridize under high stringent conditionsxe2x80x9d means that such nucleic acid molecules hybridize through complementary base pairing under conventional hybridization conditions.
The present invention relates to a polypeptide or biologically active fragments of such a polypeptides encoded by said nucleic acid molecules for the enzymatic analysis. The cytochrome P450 hydroxylase encoded by said nucleic acid molecules exhibits a C-2 hydroxylase activity that is specific to the conversions from typhasterol to castasterone and from 6-deoxotyphasterol to 6-deoxocastasterone. Further, the invention describes a polypeptide of a cytochrome P450 hydroxylase or biologically active fragments of such a polypeptide expressed in bacterial cells that exhibits the C-2 hydroxylation. The polypeptide encoded by the above-described nucleic acid molecules shares common structural and functional properties, such as molecular weights, electrophoretic mobility, chromatographic behavior, enzymatic activity, and structural and functional domains for N-terminal membrane anchoring region, the proline-rich region, and for the binding of dioxygen, heme, and steroid.
The invention also relates vectors, expression cassettes, and plasmids used in genetic engineering that contain the nucleic acid molecule as described above according to the invention.
In one aspect the present invention relates to transgenic plant cells and plants containing said nucleic acid molecule, and to experimental processes for the elucidation of other proteins involved in brassinosteroid signaling and of molecular events in the interaction of brassinosteroids and light in plant growth and development. The provision of the nucleic acid molecule according the present invention offers the potential to generate transgenic plants with a reduced or increased brassinosteroid biosynthesis leading to various physiological, morphological, and developmental changes in plants. Technical procedures for the procedures are well known to the person in the art.
With the present invention, it is possible to engineer plant growth and developmental processes, such as stem and leaf growth, in regard to the improvement of growth rate and resistance to environmental damages by introducing a brassinosteroid biosynthetic enzyme into economically important crop plants in an organ-specific manner.
Therefore, the present invention provides: 1. Nucleic acid molecules encoding a cytochrome P450 hydroxylase that catalyzes the conversions from typhasterol to castasterone and from 6-deoxotyphasterol to 6-deoxocastasterone in the brassinosteroid biosynthetic pathway in plants, comprising a nucleotide sequence as given in SEQ ID NO: 1 and 2. An Escherichia coli XL1-Blue Ddwf1 (KCTC 0857BP) containing vector pGAD4.2-1 having a nucleic acid molecule with the nucleotide sequence as given in SEQ ID NO: 1 has been deposited at Korean Collection for Type Cultures at #52, Oun-dong, Yusung-ku, Taejon 305-333, Republic of Korea, as International Depositary Authority on Aug. 28, 2000, under the Budapest Treaty.